Search PubMed⌕ Search

Biomedical subjects

D J Donaldson

Publications and source records attributed to D J Donaldson.

At least 37 records · Page 2Linked to original sources
PubMed1987

Newt epidermal cell migration in vitro and in vivo appears to involve Arg-Gly-Asp-Ser receptors.

The effect of a synthetic peptide consisting of Arg-Gly-Asp-Ser (RGDS), the amino acid sequence representing the fibroblast attachment site in fibronectin (FN), was tested on migrating newt epidermal cells. In one approach, skin explants were placed on the bottom of plastic dishes coated with human FN, human fibrinogen (FGN), human serum spreading factor (SF), or bovine type I collagen. The explants were then incubated overnight in serum-free medium with or without RGDS. In these experiments exposure to 50 micrograms ml-1 of RGDS reduced migration over FN, FGN and SF to 2-7% of control levels. Two peptides structurally dissimilar to RGDS (Val-Gly-Ser-Glu and Thr-Pro-Arg-Lys), and two that are structurally similar (Lys-Gly-Asp-Ser and Arg-Gly-Glu-Ser), had no effect on explant migration even when used at concentrations higher than 50 micrograms ml-1. Upon removal of the RGDS peptide, inhibited explants quickly recovered. In collagen-coated dishes 50 micrograms ml-1 of RGDS was much less effective than in dishes coated with the other substrates. Raising the RGDS concentration in collagen-coated dishes tenfold did not greatly increase the RGDS effect. When added to the medium bathing wounded limbs, 50 micrograms ml-1 of RGDS only moderately inhibited wound closure. This concentration of peptide, however, severely inhibited migration from skin explants in newt-plasma-coated-dishes and migration over pieces of newt-plasma-coated plastic placed under one edge of a skin wound. Increasing the RGDS concentration to 500 micrograms ml-1 resulted in almost total suppression of wound closure. Wounds exposed to this same concentration of Lys-Gly-Asp-Ser closed normally. These results indicate that newt epidermal cells possess RGDS receptors and that these receptors are involved in epidermal wound closure in vivo and in migration from skin explants onto plastic coated with FN, FGN, SF and collagen. The relative RGDS-insensitivity of wound closure in vivo and in migration from explants onto collagen may reflect in these instances the presence of a relatively high density of RGDS receptor binding sites on the substrate; the presence of RGDS receptor binding sites of relatively high affinity; or the participation of receptors other than those involved in migration over plastic coated with FN, FGN or SF.

Amino Acid Sequence↗
PubMed1986

Events in the movement of newt epidermal cells across implanted substrates.

Pieces of coverslip glass, polycarbonate filters, or coverslip plastic, coated with fibrinogen or type I collagen, were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Migratory events were then analyzed by phase contrast and electron microscopy. Phase-contrast microscopy revealed two types of lamellipodia on leading edge cells: one which was attached broadly to the cell body and one attached by a long, thin stalk. Stalkless forms were by far the most common type and we believe they provide the motive force for cell movement. Stalked-forms often moved at distinct angles to the direction of sheet movement, suggesting that they may be sensory appendages. Phase photographs of the leading edge of migrating sheet 4 hours and 8 hours after implantation showed that all cells that were on the leading edge at 4 hours continued to advance for the next 4 hours, demonstrating clearly that under these circumstances the distalmost cells do not become immobile upon contact with the substrate as others have suggested. TEM revealed that migrating sheets were modified monolayers and that regardless of proximodistal location in the sheet, and even in the intact skin adjoining a wound, each epidermal cell adjacent to the substrate puts forth a lamellipodium which underlaps the cell in front. This and the behavior of sheets as they were teased or pulled from the implant suggest strongly that all basal cells contribute to movement of the sheet by interacting with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Lectin binding to newt epidermis: fluorescent localization and effects on motility.

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Wheat germ agglutinin and concanavalin A binding during epithelial wound healing in the cornea.

It has been hypothesized that there are differences between membrane-associated glycoconjugates of wounded (migrating) epithelium and those of nonwounded (stationary) epithelium. To test this hypothesis, wheat germ agglutinin-ferritin (WGA-Fe) and concanaval in A-ferritin (Con A-Fe) binding to apical membranes of wounded and nonwounded rabbit corneal epithelia were compared. Epithelial abrasions of the superior half of the cornea were allowed to heal in vivo for six hours. Fixed corneas were then incubated with lectin-ferritin and prepared for electron microscopy. Measurements (ferritin particles per linear um of membrane) of WGA-Fe indicated that binding to leading cells (40.7 particles/um), to areas 20 to 35 cells behind the leading edge (46.5 particles/um) and to nonwounded epithelium (45.1 particles/um) from contralateral eyes were not significantly different. A competitive inhibitor of WGA, 0.2M N-acetylglucosamine, however, blocked 94 percent of WGA binding on leading cells (2.3 particles/um), while binding persisted in areas behind the leading edge (39.5 particles/um) and on nonwounded epithelium (43.6 particles/um). This indicates that leading cell surfaces have a weak affinity for WGA. Unlike WGA, Con A showed a distinct preference for leading-edge cells (33.9 particles/um) compared to nonwounded epithelium (9 particles/um). In areas 20-35 cells behind the leading edge, Con A binding was intermediate to these two extremes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosaminidase↗
PubMed1985

Location of a fibronectin domain involved in newt epidermal cell migration.

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.

Animals↗
PubMed1984

Inability of newt epidermal cells to migrate over concanavalin A-coated substrates.

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Despite the fact that concanavalin A (Con A) receptors could be demonstrated on newt epidermal cells with fluorescein isothiocyanate (FITC)-conjugated lectin, Con A-coated implants supported practically no migration, an even poorer response than the modest amount of migration that occurred on uncoated glass. Coomassie blue staining verified that the lectin formed a complete film over the glass, and peroxidase binding assays showed that even after several hours in the wound, the Con A binding sites for mannose were still available. Migration on fibrinogen-coated glass (a good migration substrate) was not affected by placing the implants next to Con A-coated implants. Thus, the failure to migrate on Con A cannot be explained by soluble Con A effects from lectin leaching off the implants. These data suggest that linkages between cell surface mannose and the substrate are not part of the strategy by which newt epidermal cells migrate.

Animals↗
PubMed1984

Epidermal cell migration on laminin-coated substrates. Comparison with other extracellular matrix and non-matrix proteins.

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for the migrating wound epithelium. Laminin, a protein that has been implicated as an epithelial-specific adhesin, was a moderately good migration substrate. Type-IV collagen, fibrinogen and fibronectin, however, were significantly better. Fetuin, myoglobin, and casein all proved to be very poor substrates, allowing practically no migration. The inability of fetuin, myoglobin, and casein to support migration is further evidence that the considerable migration that occurs on collagen (Donaldson et al. 1982) fibrinogen and fibronectin (Donaldson and Mahan 1983) and the moderate migration on laminin, is a relatively specific response to these proteins and is therefore of special significance. The fact that laminin is a poorer migration substrate than collagen, fibrinogen or fibronectin suggests that the absence of cell surface laminin that has been associated with epithelial movement in several studies (Stanley et al. 1981; Clark et al. 1982; Madri and Stenn 1982; Gulati et al. 1983) may promote motility by allowing epithelial cells to interact directly with other extracellular macromolecules.

Animals↗
PubMed1984

Influence of catecholamines on epidermal cell migration during wound closure in adult newts.

Following removal of a skin patch from each hind limb of a series of adult newts, the limbs were explanted into small dishes of Holtfreter solution containing various combinations of test drugs. Later, the amount of wound epithelium that formed on each limb was determined using a planimeter on wound tracings obtained with the aid of a drawing tube-equipped microscope. All three (-)-catecholamines tested inhibited migration with the following order of effectiveness: isoproterenol greater than epinephrine greater than norepinephrine. The effect was stereospecific with (-)-isoproterenol clearly more effective than the (+)-isomer. Propranolol, a beta antagonist, blocked the effect of (-)-isoproterenol while the alpha antagonist, phentolamine, was less effective. One hour in (-)-isoproterenol inhibited migration to the same degree as continuous exposure for the 6 hr most experiments were allowed to run. Taken together, the data suggest that catecholamines exert their effect on epidermal cell migration via beta 2 adrenergic receptors on the cell surface, a binding site which implicates cAMP in the chain of events.

Animals↗
PubMed1984

Effects of concanavalin A and cholera toxin on epidermal cAMP and migration rate during wound closure in adult newts.

Following removal of a skin patch from each hind limb of a series of adult newts, the limbs were explanted into small dishes of Holtfreter solution containing various combinations of test drugs. Later, the amount of wound epithelium that formed on each limb was determined using a planimeter on wound tracings obtained with the aid of a drawing tube-equipped microscope. Exposure of migrating cells to the plant lectin, concanavalin A (con A), lowered cyclic AMP (cAMP) levels and depressed migration. Exposure to cholera toxin and theophylline (CTX) significantly elevated cAMP levels and significantly depressed migration rate. Exposure of CTX-treated cells to con A tended to lower CTX-elevated cAMP levels while depressing the migration rate well beyond the depression caused by CTX alone. These results provide further evidence that cAMP can regulate the rate of newt epidermal cell migration. They also show that the inhibitory effect of con A on motility in these cells is independent of its effects on cAMP.

Animals↗
PubMed1983

Fibrinogen and fibronectin as substrates for epidermal cell migration during wound closure.

Pieces of glass coverslip coated with human fibronectin or human fibrinogen were implanted under one margin of a skin wound on adult newt (Notophthalmus viridescens) hind limbs. In contrast to uncoated glass or glass coated with nest serum, bovine serum or bovine serum albumin, glass treated with either fibronectin or fibrinogen supported considerable epidermal cell migration. When optimal amounts of each protein were used, the amount of migration on fibrinogen-coated glass did not differ from the amount on fibronectin-coated glass or from the amount on the wound bed. Migration on a fibronectin substrate, could be blocked by treating the substrate with an antiserum against fibronectin just prior to implantation. Similarly, migration on a fibrinogen substrate could be blocked by exposing it to an antiserum against fibrinogen. While we have yet to determine it fibrinogen and fibronectin are interacting directly with the cell surface, our observations suggest that these two proteins may play an important role in wound closure by providing a suitable substrate for epithelial cell migration.

Animals↗
PubMed1982

Epidermal cell migration on collagen and collagen-derived peptides.

Nucleopore filters coated with various genetic types of collagen and certain collagen-derived peptides were implanted under one margin of a skin wound on adult Notophthalmus viridescens (newt) hind limbs. In contrast to their behaviour on untreated filters, epidermal cells migrated readily and to equal degrees on human type I, newt type I, bovine type II, and bovine type IV collagen. Denaturation had no effect on the ability of collagen to support migration and all three cyanogen bromide peptides tested (alpha 1(I)CB3, 7 and 8) were able to support more migration than that seen on untreated filters. Glutaraldehyde-linked collagen gels supported migration but bovine serum albumin gels did not. These results show that there is no species or collagen-type specificity shown by newt epidermal cells as they migrate over collagen-coated substrates. They also demonstrate that the tertiary structure of the collagen molecule is unimportant in its ability to bind to newt epidermal cells, and that the alpha 1(I) chain has at least three, and probably many epidermal binding sites. Finally, they indicate that the improved migration on collagen is not a non-specific response to protein on the substrate.

Animals↗
PubMed1981

Epidermal cell migration during attempted closure of skin wounds in the adult newt: observations based on cytochalasin treatment and scanning electron microscopy.

Epidermal closure of skin wounds on newt limb explants was inhibited to equal degrees by cytochalasins B,D and dihydrocytochalasin B (H2CB). The cytochalasin solvent, dimethylsulfoxide (DMSO), had no effect on migration at the low concentration present in the cytochalasin and control solutions. However, a 5% DMSO solution completely blocked mobility. Wounds on limb explants and limbs in situ responded similarly to cytochalasin treatment. Inhibition of migration by H2CB was reversible even when protein synthesis was reduced by 73%. Scanning electron microscopy of wound epithelium migrating on nucleopore filters revealed extensive lamellipodia on marginal cells and the first row of submarginal cells. Cytochalasin treatment produced plications in the upper surface and free edge of the normally smooth lamellipodia. This disturbance of the free edge revealed focal adhesions with the substratum. The fact that migration was inhibition by CD and H2CB (two cytochalasins with an affinity for contractile proteins but without some of the side effects of CB) leads us to conclude that epidermal cells utilize actin or actin-like proteins during wound closure. These results increase the likelihood that tissue cells of all types, whether in vitro or in vivo, share a common biochemical basis for cell movement.

Animals↗
PubMed1980

Effect of cAMP and related compounds on newt epidermal cell migration both in vivo and in vitro.

The effect of cyclic AMP and related compounds on both in vivo and in vitro epidermal cell migration during wound closure in the adult newt was examined. Cyclic AMP (cAMP) and N6,O2'-dibutyryl cyclic AMP (db-cAMP) inhibited migration both in vivo and in vitro when used with equimolar concentrations of theophylline, an inhibitor of 3',5'-cyclic nucleotide phosphodiesterase. Neither db-cAMP nor theophylline alone inhibited migration in vivo. Adenosine 5' monophosphate (AMP), cyclic guanosine 3',5' monophosphate (cGMP) and imidazole, a potentiator of phosphodiesterase were tested in vivo and had no effect on migration. Isoproterenol and epinephrine, which are known to stimulate adenylate cyclase, inhibited migration in vitro. Experiments using the protein synthesis inhibitor, cycloheximide, suggest that cAMP could be acting partially through regulation of protein synthesis but that other factors are involved. Dibutyryl cyclic AMP and theophylline had no effect on the incorporation of 3H-leucine into protein. The inhibition of migration both in vivo and in vitro provides further evidence for a role of cAMP in the regulation of cell motility.

Animals↗
PubMed1979

Effects of x-rays on nerve-dependent (limb) and nerve-independent (jaw) regeneration in the adult newt, Notophthalmus viridescens.

The newt limb requires nerves for successful regeneration, but the jaw appears to be nerve independent. Among the current hypotheses for the regeneration-inhibitory action of X-rays is one proposing inactivation of nerves as the main cause. We decided to test this hypothesis by comparing the irradiation levels necessary for inhibition of limb and jaw regeneration. Jaws and left front limbs were exposed locally to doses of ionizing X-irradiation ranging from 250 to 2000 R at least 6 weeks prior to amputation of the jaw and both front limbs. After 90 days post-amputation all surviving animals were examined grossly for signs of regeneration. In addition, some of the controls and most of those receiving 250, 500 and 1000 R were processed for histological examination. All unirradiated limbs and jaws supported regeneration. Those exposed to 250 R also regenerated, but a third of the jaws were hypomorphic. At 500 R and above, neither jaws nor limbs regenerated. Since both systems were affected by similar doses of X-rays, it appears that nerves are not the primary X-ray target in adult newts.

Animals↗
PubMed1977

Inhibition of epidermal cell migration by concanavalin A in skin wounds of the adult newt.

Pigment cell migration was used as an indicator to study the effects of the plant lectin, concanavalin A (con A), on epidermal cell closure of skin wounds. Continuous immersion of wounded animals in 10-100 microng/ml of con A greatly slowed but did not stop epidermal cell migration. Thus, untreated animals closed wounds in 12 to 24 hours while some treated wounds were still open after three days. Removal from con A after 24 hours, allowed inhibited wounds to close faster than those left in con A. Brief (30-minute) immersion of wounded animals in con A, either before or after migration had begun, suppressed closure for four to eight hours, demonstrating that the affinity for con A persists as epidermal cells migrate. When the left forelimb of bilaterally wounded animals was immersed in con A, it caused suppression of migration only on the immersed side, indicating a local rather than systemic site of action. Mixture with its competing sugar, alpha methyl D mannoside, abolished the effects of con A. The mechanism by which this lectin suppresses epidermal cell migration is unknown but clearly involves binding of the molecule to glycoprotein or glycolipid receptors on the cell surface.

Animals↗